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Product details

SynonymsTetraspanin-20; Tspan-20; TSPAN20; UP1b; UPIb; UPK1B; Uroplakin-1b

Antibody type = Mouse monoclonal / IgG

Clone = MSVA-734M

Positive controlUrinary bladder: A strong membranous and cytoplasmic UPK1b immunostaining should be seen in the urothelium (the staining can be limited to the top cell layers or equally involve all cell layers).

Negative controlColon: UPK1b immunostaining should be absent in all cells of the colon mucosa.

Cellular localization = Cell Surface and cytoplasm

Reactivity = Human

 

Application = Immunohistochemistry
Dilution = 1:100 – 1:200
Intended Use = Research Use Only

Relevance of Antibody

Uroplakin 1B is a marker for urothelial carcinomas

Biology Behind

Uroplakin 1B (Upk1b) is a 29.6 kDa protein which is encoded by the UPK1b gene located at 3q13.3-q21. In humans, there are 4 other uroplakins (Upk 1a, -2, -3a, -3b), which assemble into dimers of Upk1a/Upk2 and Upk1b/Upk3 and eventually into heterotetramers that are deposited at the surface of the epithelial cells lining the urinary bladder, ureter, and renal pelvis. These membrane plaques are termed asymmetric unit membranes (AUMs) and serve as elastic stabilizers that prevent the bladder wall from mechanical stress and rupture during urinary bladder distension. In addition, AUMs may contribute to the regulation of membrane permeability and signal transduction, which may also impact aspects of tumorigenesis such as cellular growth and motility.

Staining Pattern in Normal Tissues

Uroplakin 1B staining pattern in Normal Tissues with antibody MSVA-734M (Images shown in our “Normal Tissue Gallery”)

Brain Cerebrum Negative.
Cerebellum Negative.
Endocrine Tissues Thyroid Negative.
Parathyroid Negative.
Adrenal gland Negative.
Pituitary gland Negative.
Respiratory system Respiratory epithelium A moderate to strong Upk1b staining occurs in a fraction of cells.
Lung
Gastrointestinal Tract Salivary glands A variable (weak to strong) Upk1b positivity can sometimes occur in some serous cells in salivary glands.
Esophagus Usually negative. Scattered Upk1b positive cells can occur.
Stomach A moderate to strong Upk1b staining is seen in superficial epithelial cells and parietal cells.
Colon Negative.
Duodenum Negative.
Rectum Negative.
Small intestine Negative.
Liver A variable (weak to strong) cytoplasmic and membranous Upk1b positivity can sometimes occur in intrahepatic bile ducts. Hepatocytes are negative.
Gallbladder A variable (weak to strong) cytoplasmic and membranous Upk1b positivity can sometimes occur in gallbladder epithelium
Pancreas Weak to moderate staining in intercalated ducts of the pancreas. Excretory ducts of the pancreas can also stain positive.
Genitourinary Kidney A variable (weak to strong) cytoplasmic and membranous Upk1b positivity can sometimes occur in the parietal layer of Bowman capsule and in few tubuli in the kidney
Urothelium Variable Upk1b staining. Equally involving all cell layers in the majority of samples but limited to the upper cell layers in some samples.
Male genital Prostate Negative.
Seminal vesicles Negative.
Testis Negative.
Epididymis Apical membrane of tall columnar cells of the epididymis may show a faint positive staining.
Female genital Breast Negative.
Uterus, ectocervix Usually negative. Scattered Upk1b positive cells can occur.
Uterus endocervix A variable (weak to strong) positivity can sometimes occur in some endocervical glands.
Uterus, endometrium A variable (weak to strong) Upk1b positivity can sometimes occur in some endometrial glands.
Fallopian Tube A variable (weak to strong) Upk1b positivity can sometimes occur in a fraction of cells in the fallopian tube.
Ovary Negative.
Placenta early A small fraction of the syncytiotrophoblast cells may stain positive.
Placenta mature A small fraction of the syncytiotrophoblast cells may stain positive.
Amnion Moderate to strong Upk1b positivity in amnion cells.
Chorion Moderate to strong Upk1b positivity in chorion cells.
Skin Epidermis Usually negative. Scattered Upk1b positive cells can occur.
Sebaceous glands Negative.
Muscle/connective Heart Negative.
Skeletal Negative.
Smooth muscle Negative.
Fat Negative.
Bone marrow/lymphoid Bone marrow Negative.
Lymph node Negative.
Spleen Negative.
Thymus Upk1b is positive in a subset of cells in corpuscles of Hassall’s. No staining of inflammatory cells.
Tonsil Upk1b is regularly positive in the surface epithelium and in the intermediate cell layer of tonsil crypt epithelium. No staining of inflammatory cells.
Remarks Staining is typically cytoplasmic and membranous.

 

These findings are largely consistent with the RNA and protein data described in the Human Protein Atlas (Tissue expression Uroplakin 1B)  All organs with documented Upk1b RNA expression (urinary bladder, kidney, prostate, gallbladder, stomach, placenta, fallopian tube, uterine cervix, tonsil) with the only exception of smooth muscle are IHC positive for MSV-734M. Given the immediate vicinity of smooth muscle to multiple Upk1b positive tissues, smooth muscle RNA positivity may represent a contamination artifact.

 

Positive control: Urinary bladder: A strong membranous and cytoplasmic UPK1b immunostaining should be seen in the urothelium (the staining can be limited to the top cell layers or equally involve all cell layers).

Negative control: Colon: UPK1b immunostaining should be absent in all cells of the colon mucosa.

 

Normal tissue gallery

Staining Pattern in Relevant Tumor Types

The TCGA database on RNA expression in cancer has described upregulation of Upk1b in a fraction of urothelial, head and neck, lung, endometrial, cervical, ovarian and renal cancers.

The TCGA findings on Uroplakin 1B RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

 

 

Cancer tissue gallery

Compatibility of Antibodies

No data available at the moment

Protocol Recommendations

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

 

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

 

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply MSVA-734M at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

 

 

 

Agilent / Dako – Autostainer Link 48

Pretreatment in PT-Link for 30 minutes at 95°C (pH high); FLEX peroxidase blocking for 5 minutes (room temperature), MSVA-734M 1:250 for 20 minutes (room temperature), FLEX+ mouse/rabbit (LINKER) for 15 minutes (room temperature), horseradish peroxidase (HRP) for 20 minutes (room temperature), FLEX DAB+Sub-Chromo for 10 minutes (room temperature), FLEX hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, and a longer incubation time of FLEX+LINKER result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

 

 

Leica – BOND RX

Dewax at 72°C for 30 seconds; Pretreatment in Bond Epitope Retrieval Solution (ER2 – EDTA pH9) for 20 minutes at 100°C; Peroxidase blocking for 5 minutes (room temperature), MSVA-734M 1:600 for 15 minutes (room temperature), Post primary (rabbit anti mouse) for 8 minutes (room temperature), Polymer (goat anti rabbit) for 8 minutes (room temperature), mixed DAB refine for 10 minutes (room temperature), hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of Post primary and or the Polymer result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

 

Roche – Ventana Discovery ULTRA

Pretreatment for 64 minutes at 100°C (pH 8,4); CM peroxidase blocking for 12 minutes (room temperature), MSVA-734M 1:150 for 20 minutes at 36°C, secondary antibody (anti-mouse HQ) for 12 minutes at 36°C, anti-HQ HRP for 12 minutes at room temperature, DAB at room temperature, hematoxylin II at room temperature for 8 minutes, bluing reagent at room temperature for 4 minutes.

These images depict staining results obtained by the protocol described above. It is of note, that the Ventana machines generally require higher antibody concentrations than other commonly used autostainers because the antibodies are automatically diluted during the procedure. Various other protocols can result in an identical result as shown above. A longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of secondary antibody and or the anti-HQ HRP result in stronger staining, potentially at the cost of more background staining.

Potential Research Applications

  • The prevalence and clinical significance of Upk1b expression in cancer is unknown.